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Abstract

Silver nanolayers were sputtered on polytetrafluoroethylene (PTFE) and subsequently
transformed into discrete nanoislands by thermal annealing. The Ag/PTFE composites
prepared under different conditions were characterized by several complementary methods
(goniometry, UV-visible spectroscopy, X-ray photoelectron spectroscopy, and atomic
force microscopy), and new data on the mechanism of Ag layer growth and Ag atom clustering
under annealing were obtained. Biocompatibility of selected Ag/PTFE composites was
studied in vitro using vascular smooth muscle cell (VSMC) cultures. Despite of the well-known inhibitory
properties of silver nanostructures towards broad spectrum of bacterial strains and
cells, it was found that very thin silver coating stimulates both adhesion and proliferation
of VSMCs.

Keywords:

Background

Nanomaterials and nanoparticles have recently received considerable attention because
of their unique properties and diverse applications in biotechnology and life science.
Nanosilver products, which have well-known antimicrobial properties, have been used
extensively in a range of medical settings [1-5].

Bactericidal properties of silver in the form of ions, nanoparticles, or composite
nanodevices based on thin Ag films have been broadly reported [6,7]. Antibacterial properties, however, are one, but not the only prerequisites for successful
integration of functional artificial materials into living tissues. Biocompatibility
and side cytotoxicity of such materials have to be considered too. Cell survival and
cell death are two major toxicity endpoints that can be rapidly and effectively measured
using in vitro experimental models employing cultured mammalian cells [8-10].

Antibacterial surface modification of biomedical materials has evolved as a potentially
effective method of preventing bacterial proliferation and biofilm formation on medical
devices [11]. Microbial colonization and biofilm formation on implanted devices represent an important
complication in, e.g., orthopedic surgery, dental surgery, or during replacement of
skin cover after severe post-traumatic conditions (burns and abrasions), and may result
in implant failure. Controlled release of antibacterial agents directly at the implant
site may represent an effective approach to treat these chronic complications [9]. Recent advances in the field of nanotechnology led scientific opinion to consider
metal nanoparticle recruitment a very promising tool to fight antibiotic-resistant
bacteria [10,11]. Among the nanomaterials, silver nanoparticles (AgNPs) have shown good inhibitory
and antimicrobial efficacy against a significant number of pathogens (such as bacteria,
viruses, yeasts, and fungal species) [12], without provoking microbial resistance [13]. Moreover, silver ions have demonstrated the capability to inhibit biofilm formation
[14]. Resistance to conventional antibiotics by pathogenic bacteria has emerged in recent
years as a major problem of public health. In order to overcome this problem, non-conventional
antimicrobial agents have been under investigation. Silver-based medical products,
ranging from bandages for wound healing to coated stents and catheters, have been
proved effective in retarding and preventing infections of a broad spectrum of bacteria
[15]. Surface proteins are probably the most Ag+-sensitive sites, and their alterations result in bacterial disruption due to structural
and severe metabolic damage. Silver ions inhibit a number of enzymatic activities
by reacting with electron donor groups, especially sulfhydryl groups [16]. In contrast to the antibacterial properties of silver (both as ions and as metallic
nanoparticles), its potential cytotoxic effects on eukaryotes have not yet been satisfactorily
elucidated [17]. However, it is clear that the potential adverse effects of AgNPs issued from their
ability to penetrate the membrane and then interfere with various metabolic pathways
of the cell [18]. Improvements in the development of non-cytotoxic, bactericidal silver-containing
products are therefore being continuously sought. In particular, increasing interest
is being shown towards the safe exploitation of silver nanotechnology in the fabrication
of bioactive biomaterials.

The main aim of this paper is to find out whether the silver nanostructures, which
are generally known for their inhibitory properties towards broad spectrum of bacterial
strains, deposited on polytetraethylfluorene (PTFE) conform to cell cultures cultivated
on this composite. For this purpose, silver-coated PTFE samples are prepared; their
properties, which are expected to affect the interaction with cells, are characterized
by different complementary experimental techniques. Special emphasis is paid to the
effects of surface morphology, chemical composition, and amount of silver. Biological
activity of silver-coated PTFE is examined in vitro on vascular smooth muscle cells (VSMCs).

Methods

Materials, Ag deposition, and thermal treatment

PTFE foil (thickness 50 μm, density 2.2 g cm−3, melting temperature Tm = 327°C), supplied by Goodfellow Cambridge Ltd. (Huntingdon, UK), was used for this
experiment. The PTFE samples were silver coated by diode sputtering using Balzers
SCD 050 device (Goodfellow Ltd.). The deposition of silver was accomplished from Ag
target (purity 99.99%), supplied by Safina a.s. (Czech Republic). The parameters of
the deposition were as follows: DC Ar plasma, gas purity 99.995%, gas flow 0.3 l s−1, pressure 5 Pa, power 20 mA, inter-electrode distance 50 mm, and sputtering time
varied from 10 to 200 s. The thermal annealing was performed immediately after Ag
deposition on air at 250°C for 1 h using thermostat Binder oven (Tuttlingen, Germany).
The annealed samples were cooled down on air to room temperature. The experiments
were performed on the samples of pristine PTFE, the Ag-coated PTFE, immediately after
the Ag deposition (as-sputtered) and after 14 days from the deposition (relaxed).
The annealed samples, relaxed for 14 days from the annealing (annealed), were used
in further experiments.

Measurement techniques

Surface wettability was characterized by contact angle (CA) measured by goniometry
using static water drop method. The analysis was performed at ten different positions
(room temperature) using distilled water (volume of water drop was 8 μl ± 0.2 μl).
The evaluation of the contact angles was performed by a three-point method using software
SeeSystem 6.3 (Advex Instruments s.r.o., Brno, Czech Republic).

The atomic concentrations of Ag (3d), O (1 s), F (1 s), and C (1 s) in Ag-coated (as-sputtered, relaxed, and annealed) PTFE were determined by X-ray
photoelectron spectroscopy (XPS) method on Omicron NanotechnologyESCAProbeP spectrometer
(Omicron NanoTechnology GmbH, Taunusstein, Germany). The analyzed area had a dimension
of 2 × 3 mm2. The X-ray source was monochromated at 1,486.7 eV, and the measurement was performed
with a step size of 0.05 eV. The spectra evaluation was carried out using CasaXPS
software (Tel Aviv, Israel).

The surface morphology and roughness of pristine, relaxed, and annealed PTFE samples
Ag coated for different deposition times were examined by atomic force microscopy
(AFM) using VEECO CP II device working in tapping mode. A phosphorous-doped silicon
probe RTESPA-CP (Veeco, Mannheim, Germany) with a spring constant of 20 to 80 N m−1 was chosen. The mean roughness value (Ra) represents the arithmetic average of the deviation from the center plane of the
sample.

Cell colonization

The interaction of pristine and Ag-coated PTFE surface (relaxed and annealed) with
the cell was studied by in vitro method. The VSMCs from the rat aorta were used in this experiment. For the studies
of cell adhesion and proliferation, the pristine and Ag-coated (sputtering times 20,
100, and 200 s) PTFE was chosen. The samples were sterilized for 1 h in ethanol (75%)
and air dried before the experiment. The samples were inserted into 12-well plates
(TPP, Trasadingen, Switzerland) and seeded with VSMCs with the density of 17,000 cells
cm−2 into 3 ml of Dulbecoo’s modified Eagle’s essential medium (Sigma, USA) supplemented
with 10% fetal bovine serum (Sebak GmbH, Germany). The cells were cultured for 1,
2, 5, and 7 days at 37°C in a humidified atmosphere containing 5% CO2.

For cell number analysis and cell distribution on sample surface, the method of randomly
chosen fields was chosen. On the first, second, fifth, and seventh day from seeding,
the cells were rinsed with phosphate-buffered saline (Sigma), fixed for 45 min in
75% cold ethanol (at 20°C), and stained (1 h) with a combination of the fluorescence
dyes. Texas Red C2-maleimide (Invitrogen Ltd., Renfrew, UK) was used for dying the cell membrane. The
cell nuclei were visualized using Hoechst #33342 (Sigma). The fluorescent microscope
Olympus IX-51 (Evropská, Czech Republic) with digital camera DP-70 was used for the
creation of the 20 photographs from different positions of the samples. The number
of cells was determined using NIS-Elements AR3.0 software (Nikon, Melville, NY).

Results and discussion

Since the cell adhesion and proliferation are strongly affected by chemical composition,
surface morphology, wettability, and other physicochemical properties of underlying
carrier, the silver/PTFE composites prepared under different conditions were characterized
by various complementary analytical methods.

Contact angle measurement

The dependence of the CA of silver-coated PTFE on the silver sputtering time from
10 to 200 s is shown in Figure 1 and compared with that of pristine PTFE (CA = 110.5° ± 2.0°). The contact angle was
determined immediately after silver deposition (as-deposited), after 14 days from
the silver deposition (relaxed), and on annealed and relaxed samples (annealed).

Figure 1.Dependence of contact angle on sputtering time for pristine (deposition time 0 s)
and silver-coated PTFE. Contact angle was determined immediately after Ag deposition (as-sputtered), after
14 days from the Ag deposition (relaxed), and on annealed and relaxed samples (annealed).

The deposition of Ag layer onto PTFE results in significant CA decrease (i.e., increase
of wettability), due to pronounced masking effect of the Ag layer. This decrease is
most pronounced in the case of the thickest Ag coatings (sputtering time > 160 s),
for which the creation of fully continuous coverage is expected in accordance with
previous work [19]. For the as-deposited samples, three distinguishable regions are seen on the dependence
of CA on the sputtering time. In the first region, the contact angle is a decreasing
function of sputtering time (deposition time 10 to 40 s). The second region is characterized
by nearly constant, within experimental error, CA value of about 92° (sputtering times
40 to 140 s). In the third region (sputtering time > 160 s), the contact angle falls
down to the mean value of about 72°. This decline is due to the formation of continuous
Ag layer. The annealed samples exhibit entirely different dependence of CA on the
sputtering time. The annealing of ultrathin Ag layers results in slight decrease of
CA for sputtering times of 10 to 30 s. The initial drop is followed by a gradual increase
of CA up to the value close to that of pristine PTFE. For thicker layers (sputtering
times > 80 s), the CA remains practically constant, reflecting the fact that the post-deposition
annealing leads to the coalescence of the Ag atoms into discrete islands (see Figure 2 and Table 1) and partial uncovering of the PTFE surface. Anomalous drop of contact angle at the
initial stage of deposition is probably due to the disposition of silver to react
with oxygen from ambient atmosphere (see, e.g., [20]). This phenomenon is particularly pronounced in tiny Ag structures [21]. Oxygen-rich compounds increase the sample wettability (see also Table 1; Ag/O ratio becomes lower for thin annealed layers).

UV–vis spectroscopy

UV–vis absorption spectra of relaxed (A) and annealed (B) samples are shown in Figure 3. As expected, the absorbance increases with increasing deposition time as the Ag
layer becomes thicker. The spectra of the annealed samples exhibit distinctive narrow
absorption peak at about 400 nm, corresponding to the surface plasmon resonance (SPR)
in silver nanostructures. It is well known that the position and shape of the SPR
peak is closely related to the nanostructure shape and to the surrounding medium [22,23]. The appearance of absorption peak after annealing indicates the formation of discontinuous
Ag clusters of hummock-like shape (see Figure 2) homogeneously distributed over the PTFE surface [24]. The absorption band corresponding to the bounded plasma resonance in the metal nanostructures
is slightly shifted to longer wavelengths when the cluster density increases. Moreover,
as the silver layer becomes thicker, the absorption band broadens due to wider distribution
of the cluster size. The spectra of the as-deposited samples (Figure 3A) with deposition times below 30 s possess only weak SPR peak. In this case, the
SPR peak is widespread and hardly identifiable because of insufficient separation
of fundamental building blocks (clusters) of silver layer in the initial stage of
the layer growth, where the formation of discontinuous but interconnected Ag coating
is expected [19].

Chemical composition

Besides the wettability, the chemical composition of the sample surface plays essential
role in material biocompatibility [25,26]. Moreover, the elemental composition is closely linked to the wettability. The results
from the XPS elemental analysis of the Ag/PTFE composites are summarized in Table 1. The average information depth of the present XPS measurement is limited to approximately
8 to 10 atomic surface layers. One can see that with ongoing deposition, the concentration
of silver increases, while the fluorine content decreases and becomes undetectable
on the sample sputtered for 200 s. The decrease is due to the increasing masking effect
of the growing Ag layer which at last becomes homogeneous and continuous. On the other
hand, with decreasing thickness of Ag layer, its masking effect gradually declines,
e.g., because of the appearance of cracks and discontinuities in the layer, and the
chemical structure of the underlying PTFE becomes visible in the XPS spectra. For
the sputtering time of 20 s, the measured fluorine concentration of 37.3 at.% is close
to that of the pristine PTFE. The F/C ratio of silver-sputtered samples is markedly
different from that of the pristine PTFE (F/C = 2:1) and may be due to the ability
of silver to attract hydrocarbon contaminants from ambient atmosphere [27]. The thicker the sputtered layer, the lower the F/C ratio. This effect is most pronounced
in the case of the thickest Ag layer (200-s sputtering time), where fluorine is not
detected because of the masking effect of the silver coating. However, the concentration
of carbon is still notable (54 at.%) in this case. The origin of carbon may completely
be attributed to the contamination with hydrocarbons and other carbon-rich compounds
from ambient atmosphere.

XPS data (Table 1) also elucidate the processes in the course of the sample relaxation. During the
14 days of relaxation, the surface chemical composition changes significantly. A gradual
decrease of the detected silver content, compared to that of the as-sputtered samples,
occurs as a consequence of the tendency to minimize surface energy at the metal-polymer
interface. This phenomenon has been frequently observed especially in the case of
plasma-treated polymers, where oxygen-containing groups reorient towards polymer volume
in order to reduce surface energy in the contact with ambient atmosphere [28]. Thus, the relaxation leads to segregation on the metal-polymer interface and boarding
of cracks in the silver coating (Table 1, increase of fluorine content). This process favorably affects the surface wettability
which finally stabilizes at a constant level (Figure 1). However, there are other concurrent processes that make the simple and straightforward
explanation of the observed phenomena difficult (e.g., anomalous decrease of fluorine
content for deposition time of 20 s, Table 1). This may particularly be caused by random, uncontrollable adsorption of hydrocarbons
from ambient atmosphere during the relaxation process (see decrease of oxygen content
at 100 and 200 s deposition times, Table 1). Since the changes of the surface properties (chemical composition and wettability)
during the sample relaxation lead to the stabilization of the sample properties (Figure 1) and, in this way, to the improvement of reproducibility of further experiments on
biocompatibility, these were performed on relaxed samples only.

Dramatic change in the surface chemistry occurs after the annealing (Table 1). Sharp drop in silver concentration for the samples sputtered for 100 and 200 s
is caused by intensive coalescence of the Ag atoms into island-like formations (also
Figure 2). This phenomenon is most pronounced for the sample sputtered for 20 s, in which
no Ag is detected by the XPS method. With proceeding Ag coalescence, the F concentration
increases dramatically as the original PTFE surface becomes uncovered, and simultaneously
the measured F/O ratio approaches the value of pristine PTFE (F/O = 2:1). The lack
of oxygen after the annealing may be attributed to the well-described effect of desorption
of oxygen-rich contaminated product and reduction of oxidized silver [27].

Surface morphology and roughness

Surface roughness and morphology of the substrates play a crucial role in adhesion
and proliferation of cells [29,30]. AFM images of pristine, relaxed, and annealed silver-coated PTFE are shown in Figure 2 together with the corresponding values of surface roughness Ra (Table 2). For the sake of comparison, appropriate vertical scales were chosen for the particular
images. The surface roughness of the relaxed Ag films decreases with increasing deposition
time (Table 2), the decrease reflecting the layer growth mechanism [31]. During the initial stage of the layer growth, isolated silver islands (separated
clusters) are formed, and the surface roughness increases compared to that of the
pristine polymer. Longer deposition leads to the formation of interconnections between
clusters, and the deposited layer becomes more homogeneous and uniform (see Table 1). This process is accompanied by gradual decrease of the surface roughness. Subsequent
annealing results in pronounced change in the surface morphology. Annealing leads
to silver coalescence and formation of hummock-like structures which are easily identifiable
in the AFM images of samples which are Ag coated for different deposition times (Figure 2 annealed). This coalescence is due to the accelerated diffusion of Ag atoms at elevated
temperature, and the formerly continuous Ag layer transforms into an island-like structure.
The dimension of such structures is a function of the thickness of the Ag layer prior
to annealing. The decomposition of the dense film into particles and clusters, known
as solid-state dewetting [32], is driven by the minimization of surface energy. It should be noted that metals
(e.g., gold) in the form of nanosized structures (rods, disks, and clusters) melt
at lower temperatures than those in bulk materials. Those melting temperatures fall
down to values between 300°C and 400°C, depending on the size and shape of the nanostructures
[33,34].

Cell adhesion and proliferation

The adhesion and proliferation of VSMCs from the rat aorta were studied in vitro on the as-sputtered and annealed samples, both relaxed for 14 days. Cell adhesion
is the first stage of cell-material interaction and occurs during the first 24 h from
cell seeding. This process leads to the anchoring of the cells through specific binding
interactions for a particular surface. Adhesion stage is controlled by the current
state of the substrate surface. The second phase of the cell interaction is so called
lag phase. It is the time required for cells to adapt to the new environment, and
it takes approximately 24 to 48 h. After overcoming this stage, the cells can start
to growth, spread, and proliferate.

The degree of cell adhesion was determined as the number of cells found on the sample
surface after 24 h from seeding. The dependence of the adhered VSMCs on the Ag sputtering
time is shown in Figure 4A,B for relaxed and annealed samples. For comparison, the result for pristine PTFE
(sputtering time 0 s) is also shown. From Figure 4A (as sputtered and relaxed samples) it is obvious that the presence of Ag coating
has a positive effect on cell adhesion. The number of VSMCs found on the Ag-coated
samples was comparable (3,150 ± 480 cells cm−2) for different sputtering times, whereas the adhesion on pristine PTFE was found
to be very low (490 ± 280 cells cm−2). This result is rather unexpected since it is known that in general, the presence
of nanosized Ag on tissue carriers has a negative effect on cell growth. In the case
of the annealed samples (see Figure 4B), the situation is rather different. The highest increase of the adhered cells (2,830
cells cm−2) was observed on the sample sputtered for 20 s, while the cell adhesion on pristine
PTFE and the samples Ag sputtered for longer deposition times (100 and 200 s) was
minimal (Figure 4B). It is probably due to both lower wettability (caused by desorption of oxygen-rich
compounds during annealing) and higher roughness of the samples.

Figure 4.The number of VSMC dependence on silver sputtering time. The dependence of number of VSMCs on silver sputtering time for as-sputtered (A) and annealed (B) samples for different cultivation periods (first, second, fifth, and seventh days).

Proliferation was determined as the number of VSMCs found on the samples after 2,
5, and 7 days from seeding (see Figure 4). The most significant changes were observed after the seventh day of cultivation.
On the samples deposited for 20 s, a high cell number was found (72,650 ± 24,700 cells
cm−2 for as-deposited and 29,300 ± 19,500 cells cm−2 for annealed samples). Higher proliferation on these samples occurred, owing to the
formation of discontinuous metal layer and the favorable combination of the two factors,
surface roughness and wettability. This result confirms the well-known fact that biocompatibility
depends not only on the chemical composition but also on the surface properties such
as the aforementioned wettability and roughness [35]. The low contact angle (high wettability), presence of oxygen in the surface layer,
and rough surface of the substrate are prerequisites for successful VSMC adhesion.
Thus, the difference in the number of proliferated cells between annealed and relaxed
samples can be attributed to the different elemental compositions of the surface layer
and resulting different wettability. From Figure 4A,B, it is evident that the cell proliferation on the other samples, sputtered for
longer times, is very low. Sputtering for longer times (100 and 200 s), which leads
to the formation of homogenous and continuous metal coverage, has a negative effect
on cell interaction from the long-term point of view.

The above results are illustrated on the photographs of the adhered (first day from
seeding) and proliferated (seventh day from seeding) cells on the relaxed and annealed
samples (Figure 5). The cells cultivated for 24 h are equally distributed on the surface. The cells
on the samples that are as-sputtered for 20 s and those on subsequently annealed samples
start spreading, and their adhesion increases; however, the cells on the samples sputtered
for 200 s and coated completely with silver stay small and round shaped. After 7 days
from the seeding, the cells on the samples sputtered for 20 s are numerous and evenly
distributed over the sample surface. The cell proliferation on the samples sputtered
for 200 s is much worse. In the case of the as-sputtered layer, the silver forms homogenous
coverage, completely shading the original polymer surface. After annealing of the
thicker Ag layer, a dramatic coalescence of silver into distinctive hummock-like structures
takes place, the latter being high enough to prevent a contact between polymer substrate
and adhered cells.

Figure 5.Photographs of adhered and proliferated VSMCs. Photographs of VSMCs adhered (first day) and proliferated (seventh day) on Ag-coated
PTFE with different deposition times (20 and 200 s) for as-sputtered and annealed
samples.

Conclusions

The properties of silver layers sputtered on PTFE for different times and their changes
under annealing were studied by different methods. The biocompatibility of the samples
prepared under different conditions was examined in vitro experiments with vascular smooth muscle cells. Relations between physicochemical
properties of silver layers and their biocompatibility were found. Coating with silver
leads to an increase of surface wettability, which is further affected by oxidized
structures adsorbed by the sample surface. With the increasing thickness of the silver
layer, an increase of the oxygen concentration is also observed which is explained
by high affinity of silver to oxygen and oxidized structures. Post-deposition annealing
leads to the dramatic change in morphology of the deposited silver layer and silver
coalescence. Formerly, continuous or semicontinuous Ag layers are transformed into
discontinuous ones, consisting of discrete hummock-like structures. In this way, the
surface of PTFE may be partly uncovered by annealing. UV–vis absorption increases
with increasing deposition time as the Ag layer becomes thicker. The UV–vis spectra
of the annealed samples exhibit distinctive narrow absorption peak at about 400 nm,
corresponding to the SPR in the silver nanostructures. The detailed characterization
of Ag/PTFE composites, prepared under different conditions, was a prerequisite for
the next experiments on their biocompatibility. The most important contribution of
this work is the finding that the silver nanostructures, which are generally known
for their inhibitory properties towards broad spectrum of bacterial strains and cells,
under such specific conditions conform to cell cultures cultivated on PTFE support
coated with those nanostructures. Best biocompatibility, cell adhesion, and proliferation
were exhibited by the PTFE samples Ag sputtered for 20 s. Post-deposition annealing
does not improve the sample biocompatibility. Increased biocompatibility of the samples
coated with thin Ag layer is explained by favorable combination of the sample surface
morphology and higher wettability. The biocompatibility of the samples sputtered for
longer times and coated with thicker Ag layer is miserable. Last but not least, the
results obtained by different diagnostic techniques on Ag/PTFE composites are of importance
for better understanding of the growth mechanism of metal layer on polymer substrates
and their behavior under annealing.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

JS conceived of the study, carried out the thickness and AFM measurements. He designed
and drafted the study. MP carried out and evaluated the contact angle and UV–vis measurements.
NSK performed the cell adhesion and proliferation measurements together with its evaluation.
ZK participated in the determination of the chemical composition. VS participated
in the design of the study and its coordination. All authors read and approved the
final manuscript.

Acknowledgement

Financial support of this work from the GACR project nos. P108/11/P337 and P108/10/1106
is gratefully acknowledged.